Antibacterial Activity of Aspergillus Isolated from Different Algerian Ecosystems

Vol. 16(32), pp. 1699-1704, 9 August, 2017 DOI: 10.5897/AJB2017.16086 Article Number: 28412E265692 ISSN 1684-5315 African Journal of Biotechnology Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/AJB Full Length Research Paper Antibacterial activity of Aspergillus isolated from different Algerian ecosystems Bramki Amina1*, Ghorri Sana1, Jaouani Atef2, Dehimat Laid1 and Kacem Chaouche Noreddine1 1Laboratory of Mycology, Biotechnology and Microbial Activity, University of Mentouri Brothers- Constantine, P.O. Box, 325 Ain El Bey Way, Constantine, Algeria. 2Laboratory of Microorganisms and Active Biomolecules, University of Tunis El Manar, Campus Farhat Hached, B.P. no. 94 - Rommana, Tunis 1068, Tunisia. Received 26 May, 2017; Accepted 4 August, 2017 Thirty two strains of Aspergillus genus were isolated from soil samples obtained from particular ecosystems: Laghouat endowed with a desert climate and Teleghma with a warm and temperate climate. Based on the morphological aspect, this collection was subdivided into ten phenotypic groups. This identification was confirmed by molecular analyzes using a molecular marker of the genu ribosomal 18s. This marker will allow us to associate our sequences with those of known organisms. In order to discover new antibiotic molecules, the antibacterial activity was performed against two Gram positive bacteria: Staphylococcus aureus and Bacillus subtilis and also two Gram-negative bacteria: Escherichia coli and Pseudomonas aeroginosa, using two different techniques: Agar cylinders and disks technique. The results show that the fungal species have an activity against at least one test bacterium. The Gram positive bacteria were the most affected, where the averages of the inhibition zones reach 34.33 mm. However, Gram-negative bacteria showed less important results from 0 to 12.00 mm. It is recorded that the antibacterial activity was studied for the first time in the following two species: Aspergillus niveus and Aspergillus wentii. Furthermore, an indepth study is underway on bioguided fractionation, which would identify individual components and lead to the isolation of the active ingredient. Key words: Aspergillus, particular ecosystems, antibacterial activity. INTRODUCTION The massive use of antibiotics has not led to the making multi-resistant strains to be responsible for elimination of infections, but has made microbes resist serious infections. In recent years, bacterial resistance to these antibiotics by exerting a selection pressure which antibiotics has become a worrying global phenomenon favors the emergence of resistance genes in bacteria, (Courvalin and Philippon, 1990; Bevilacqua, 2011). Due *Corresponding author. E-mail: [email protected]. Tel: +213 6 69 43 31 06. Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 1700 Afr. J. Biotechnol. to the increase in infections involving multi-resistant followed by successive subcultures in the same isolation medium bacteria, the need for new effective antibacterial (Boudoudou et al., 2009), and they were preserved in the form of treatments is more and more imperious (Savin, 2014). spores in a glycerol/saline solution of 30% at -20°C (Botton et al., 1990). For this, the biodiversity is exploited. The isolation of strains from rarely exploited ecosystems allows the discovery of strains that may have a high potential or Morphological identification unexploited production. In addition, the use of antibiotic- resistant bacteria as germs tests can lead to the The strain identification was performed by macroscopic observation (growth rate, colonies color and color variation over time, colonies discovery of effective molecules which may be new upside color, surface texture, etc) and microscopic characters (Boughachiche et al., 2012). In this field, fungi have been (mycelium, conidiophores, conidiogenesis, conidia, etc) (Botton et widely studied. Of a total of 10700 antibiotics described al., 1990; Chabasse et al., 2002, 2008). for the entire living world, about 1600 are from fungi (Botton et al., 1990). Molecular identification Aspergillus species are ubiquitous molds, widely distributed in nature (Bennett et al., 2010). From an DNA extraction and quantification economic view, the species of this genus have a great ecological and medical importance. The production of First, a quantity of mycelium (≈ 100 mg) was mashed in the extracellular and intracellular secondary metabolites by presence of 500 µl of lysis buffer (400 Mm Tris/HCl, 60 Mm EDTA, several species of this genus was detected in Aspergillus 150 mM NaCl, 1% SDS, 2 ml H2O ultrapure). The mixture was left to rest for 10 min and then 150 µl of potassium acetate was added aculeatus, Aspergillus ochraceus and Aspergillus terreus. (pH 4.8, solution prepared from 60 ml of a potassium acetate The latter are considered to be the highest producers of solution 5 M, 11.5 ml glacial acetic acid and 28.5 ml milliQ water). extracellular secondary metabolites (Youcef-Ali, 2014). After that, the tube was homogenized using a vortex, and Indeed, Aspergillus are known for their ability to produce centrifuged for 10 min at 10000 rpm, and the supernatant was antibacterial substances such as A. flavus used in the transferred to another tube of 1.5 ml volume on which an equal manufacturing of aspergillic acid, A. ochraceus used in volume of isopropanol was added. Then, the tube was agitated by inversion (10 times) and the manufacturing of penicillic acid and A. Fumigatus incubated at 20°C overnight. After incubation and centrifugation used in the manufacturing of fumagillin (Botton et al., (10000 rpm for 10 min), the supernatant was thrown and the DNA 1990; Taniwaki et al., 2003; Abdelaziz, 2006). pellet was rinsed twice with 300 µl of 70% ethanol. After This study aimed to show the antibacterial activity of centrifugation (10000 rpm for 10 min), the supernatant was Aspergillus strains isolated from different ecosystems and removed and dried in the hood for 2 h. The DNA was dissolved in producers of active metabolites used against Gram 50 µl of TE (Tris-EDTA). The DNA assay was realized by spectrophotometer (Thermo positive and negative resistant bacteria through different Scientific NanoDrop 2000) at two different wavelengths: the methods. absorption wavelength of nucleic acids (260 nm), and the absorption wavelength of proteins (280 nm). Then the DNA samples were stored at -20°C for further use (El Khoury, 2007). MATERIALS AND METHODS Sampling PCR amplification of internal transcribed spacer (ITS) regions of rDNA This work deals with the isolation of Aspergillus from soil. For this purpose, the sampling was done in two different Algerian regions The Internal Transcribed Spacer (ITS) region of the ribosomal DNA namely: Laghouat (located 400 km in south Algeria at 750 m of was amplified with universal primers ITS1 (5’- altitude) and Teleghma (located in north - eastern Algeria). TCGGTAGGTGAACCTGCGG-3’) and ITS4 (5’- To do this, 100 g of soil was sampled up to 20 cm deep after TCCTCCGCTTATTGATATGC-3’) (White et al., 1990). For isolates, discarding the first three centimeters of soil, and then deposited in PCR was performed in a total reaction volume of 25 μl containing 5 glass bottles under strict aseptic conditions. Mycological analysis μl of 5X buffer (Promega), 1 μl of dNTP (20 mM), 1.5 μl of MgCl2 was done on arrival at the laboratory (Pochon, 1964; Almi et al., (25 mM), 0.25 U (5 U/μl) Taq DNA polymerase, 2 μl of each primer 2015). (20 pmoles/μl), 11.25 µl of ultrapure H2O, and 2 μl of genomic DNA (50 ng/μl). The amplification program included an initial denaturation at 95°C Isolation for 3 min, followed by 35 cycles of denaturation at 98°C (15 s), annealing at 59°C (60 s), extension at 72°C (120 s) and a final The isolation was performed by the suspension - dilution method extension period of 10 min at 72°C in a Biometra thermal cycler (Davet, 1996; Davet and Rouxel, 1997). Decimal dilutions were (Germany). prepared from 10-1 to 10-6. Then, 0.1 ml of each suspension is rolling out on the surface of a potato dextrose agar (PDA: 200 g of potato, 20 g sugar, 20 g agar and 1000 ml distilled water) medium. Sequencing Three dishes were prepared for each dilution, and the plates were incubated at 28°C for 6 days. After an electrophoresis on 1.5% agarose gel of the amplification The proliferation of bacteria was prevented by adding an products, an enzymatic sequencing of DNA fragments was antibiotic, streptomycin (5 mg/l) in the culture medium (Botton et al., performed according to the method of incorporation of 1990). After the appearance of fungal colonies, these were purified dideoxynucleotide terminators system (Sanger et al., 1977). After Bramki et al. 1701 purification and quantitation, a precise aliquot of DNA was used as technique, DMSO was used as negative control. It should be noted a template of an enzymatic amplification reaction in the presence of that four repetitions were performed for each technique. And the dideoxynucleotides (ddNTP's) labeled with Big Dye. diameters of the inhibition zones were measured in millimeter. Sequence comparison with the data bank RESULTS AND DISCUSSION Sequence comparison with those of the databases was performed using the BLAST algorithm (Altschul et al., 1997). The alignments Screening has always been the key to achieving new of the nucleotide sequences were realized with the software antibacterial molecules. Although its performance has Chromas (Larkin et al., 2007). been reduced in recent years, it is been practiced till date in many laboratories. They have endeavored to diversify Antibacterial activity of isolated Aspergillus species the sources of microorganisms by developing selection methods that favor new species (Le Berre and All the isolated species were tested for their antibacterial activity. Ramousse, 2003). For that purpose, two techniques were used: the agar cylinders and For this purpose, our work focuses on the research of disc technique.

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